Regulation of intracellular protein trafficking is one of the central issues in cell biology. Extracellular proteins are delivered to cells via endocytosis, in which the macromolecules are sorted to their ultimate intracellular destination, often the lysosome for proteolysis, whereas the major proteolytic fate of cytoplasmic (and some membrane) proteins is via the ubiquitin pathway. During the past grant period, we demonstrated a linkage between the endosomallysosomal pathway and the ubiquitin proteolytic pathway. Thus, our long term goals are to elucidate the molecular mechanisms responsible for the cell biology of intracellular protein sorting and degradation. Our most recent studies have focused on tow aspects of the ubiquitin pathway, namely the role of the first and rate-limiting step (the ubiquitin activating enzyme, E1) and identification of new enzymes of this pathway which confer recognition for degradation on a host of regulated cellular proteins. During these studies we demonstrated the presence of the ubiquitin system within the cell nucleus and its regulation during the cell cycle. This raises several questions focused on the role of the ubiquitin system in the nucleus. Thus, the specific aims of the present proposal are (1) to elucidate the elements and regulatory mechanisms which govern the nuclear ubiquitin proteolytic pathway and (2) to define the structure and function of new factors of the ubiquitin system which govern the degradation of specific regulatory proteins which function within the nucleus. Our approach to these questions will take advantage of biochemical, cell and molecular biological approaches, including use of purified components, antibodies, cell lines harboring mutations in the pathway, in vitro reconstituted systems. These studies taken together will advance our understanding of the cellular biology of intracellular protein degradation.